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 CHAPTER 10
ANALYZING GENES AND GENOMES
© 2009 Garland Science Publishing

10-1 Recombinant DNA technologies involve techniques that permit the creation of
custom-made DNA molecules that can be introduced back into living organisms.
These technologies were first developed in the ______.
(a) 1930s
(b) 1950s
(c) 1970s
(d) 1990s

Manipulating and Analyzing DNA Molecules

10-2 Which of the following statements about restriction nucleases is false?

(a) A reproducible set of DNA fragments will be produced every time a
restriction nuclease digests a known piece of DNA.
(b) Restriction nucleases recognize specific sequences on single-stranded
DNA.
(c) Some bacteria use restriction nucleases as protection from foreign DNA.
(d) Some restriction nucleases cut in a staggered fashion, leaving short single-
stranded regions of DNA at the ends of the cut molecule.

10-3 You have purified DNA from your recently deceased goldfish. Which of the
following restriction nucleases would you use if you wanted to end up with DNA
fragments with an average size of 70 kilobase pairs (kb) after complete digestion
of the DNA? The recognition sequence for each enzyme is indicated in the right-
hand column.
(a) Sau3AI GATC
(b) BamHI GGATCC
(c) NotI GCGGCCGC
(d) XzaI GAAGGATCCTTC

10-4 You have a piece of circular DNA that can be cut by the restriction nucleases
XhoI and SmaI, as indicated in Figure Q10-4.

Figure Q10-4
 If you were to cut this circular piece of DNA with both XhoI and SmaI, how
many fragments of DNA would you end up with?
(a) 1
(b) 2
(c) 3
(d) 4

10-5 You have a piece of circular DNA that can be cut by the restriction nucleases
EcoRI, HindIII, and NotI, as indicated in Figure Q10-5.

Figure Q10-5

Which of the following statements is false?

(a) One piece of DNA will be obtained when this DNA is cut by NotI.
(b) A piece of DNA that cannot be cut by EcoRI will be obtained by cutting
this DNA with both NotI and HindIII.
(c) Two DNA fragments that cannot be cut by HindIII will be obtained when
this DNA is cut by EcoRI and NotI.
(d) Two DNA fragments of unequal size will be created when this DNA is cut
by both HindIII and EcoRI.

10-6 You have accidentally torn the labels off two tubes, each containing a different
plasmid, and now do not know which plasmid is in which tube. Fortunately, you
have restriction maps for both plasmids, shown in Figure Q10-6. You have the
opportunity to test just one sample from one of your tubes. You have equipment
for agarose-gel electrophoresis, a standard set of DNA size markers, and the
necessary restriction enzymes.

Figure Q10-6
 A. Outline briefly the experiment you would do to determine which plasmid
is in which tube.
B. Which restriction enzyme or combination of restriction enzymes would
you use in this experiment?

10-7 Assume that defects in a hypothetical gene X have been linked to antisocial
behavior. Two copies of a defective gene X predispose a child to bad behavior
from childhood, whereas a single copy of the gene seems to produce no symptoms
until adulthood. Because early treatment can counteract the effects of the gene, a
program of voluntary genetic testing is being performed with delinquent
prospective parents. Charles S. and Caril Ann F. have been arrested on charges of
robbery and assault, and Caril Ann is pregnant with Charles’s child. You obtain
DNA samples from Charles, Caril Ann, and the fetus. You digest these samples
with NotI and use these samples to perform two Southern blots, which you probe
with two different oligonucleotide probes, A and B, that hybridize to different
parts of the normal gene X, as shown in Figure Q10-7A. Your results are shown
in Figure 10-7B.

Figure Q10-7

A. Which of the three individuals have defects in gene X?

B. Which individuals have a single defective gene and which have two
defective copies of the gene?
C. Indicate the nature (single-base-pair mutation or deletion) and location of
each individual’s defects on gene X.

10-8 Figure Q10-8 shows a restriction map of a piece of DNA containing your favorite
gene. The arrow indicates the position and orientation of the gene in the DNA. In
part (B) of the figure are enlargements showing the portions of the DNA whose
 sequences have been used to make oligonucleotide probes A, B, C, and D. Which
of the oligonucleotides can be used to detect the gene in each of the following?

Figure 10-8

A. A Southern blot of genomic DNA cut with HindIII.

B. A Northern blot.

10-9 During gel electrophoresis, DNA fragments _______________________.

(a) travel through a matrix containing a microscopic network of pores
(b) migrate toward a negatively charged electrode
(c) can be visualized without stains or labels
(d) are separated on the basis of their sequence

10-10 A double-stranded DNA molecule can be separated into single strands by heating
it to 90°C because _______________________.
(a) heat disrupts the hydrogen bonds holding the sugar-phosphate backbone
together
(b) DNA is negatively charged
(c) heat disrupts hydrogen bonding between complementary nucleotides
(d) DNA is positively charged

10-11 You are interested in a single-stranded DNA molecule that contains the following
sequence:

5′- ..... GATTGCAT .... -3′

Which molecule can be used as a probe that will hybridize to your sequence of
interest?
(a) 5′-GATTGCAT-3′
(b) 5′-TACGTTAG-3′
(c) 5′-CTAACGTA-3′
(d) 5′-ATGCAATC-3′

DNA Cloning

10-12 For each of the following sentences, fill in the blanks with the best word or phrase
selected from the list below. Not all words or phrases will be used; use each word
or phrase only once.
 A nuclease hydrolyzes the __________________ bonds in a
nucleic acid. Nucleases that cut DNA only at specific short
sequences are known as __________________. DNA composed of
sequences from different sources is known as
__________________. __________________ can be used to
separate DNA fragments of different sizes. Millions of copies of a
DNA sequence can be made entirely in vitro by the
__________________ technique.

DNA sequencing phosphodiester

endonucleases polymerase chain reaction
exonucleases recombinant DNA
gel electrophoresis restriction nucleases
hydrogen ribonucleases
nucleic acid hybridization

10-13 For each of the following sentences, fill in the blanks with the best word or phrase
selected from the list below. Not all words or phrases will be used; use each word
or phrase only once.

Two fragments of DNA can be joined together by

__________________. Restriction enzymes that cut DNA straight
across the double helix produce fragments of DNA with
__________________. A fragment of DNA is inserted into a
__________________ in order to be cloned in bacteria. A
__________________ library contains a collection of DNA clones
derived from mRNAs. A __________________ library contains a
collection of DNA clones derived from chromosomal DNA.

blunt ends DNA polymerase RNA

cDNA genomic staggered ends
DNA ligase probe vector

10-14 Name three features that a cloning vector for use in bacteria must contain. Explain
your answers.

10-15 Figure Q10-15 shows the cleavage site of several restriction nucleases.

Figure Q10-15
 You cut a vector using the PclI restriction nuclease. Which of the following
restriction nucleases will generate a fragment that can be ligated into this cut
vector with the addition of only ligase and ATP?
(a) HindIII
(b) NcoI
(c) MmeI
(d) NspV

10-16 Figure Q10-16 depicts a strategy by which a DNA fragment produced by cutting
with the EcoRI restriction nuclease can be joined to a DNA fragment produced by
cutting DNA with the HaeIII restriction nuclease.

Figure Q10-16

Note that cutting DNA with EcoRI produces a staggered end, whereas cutting
DNA with HaeIII produces a blunt end. Why must polymerase be added in this
reaction?
(a) Polymerase will fill in the staggered end to create a blunt end.
(b) Polymerase is needed to seal nicks in the DNA backbone.
(c) Polymerase will add nucleotides to the end produced by the HaeIII
restriction nuclease.
(d) Without polymerase, there will not be enough energy for the reaction to
proceed.

10-17 DNA can be introduced into bacteria by a mechanism called ____________.

(a) transcription
(b) ligation
(c) replication
(d) transformation

10-18 Which of the following statements about gel-transfer hybridization (or Southern
blotting) is false?
(a) This technique involves the transfer of DNA molecules from gel onto
nitrocellulose paper or nylon paper.
(b) In this technique, single-stranded DNA is separated by electrophoresis.
(c) A labeled DNA probe binds to the DNA by hybridization.
(d) The DNA that is separated on a gel is not labeled.
10-19 Figure Q10-19 shows the recognition sequences and sites of cleavage for the
restriction enzymes SalI, XhoI, PstI, and SmaI, and also a plasmid with the sites
of cleavage for these enzymes marked.

Figure Q10-19

A. After which of the five treatments listed below can the plasmid shown in
Figure Q10-19 re-form into a circle simply by treating it with DNA ligase?
Assume that after treatment any small pieces of DNA are removed, and it
is the larger portion of plasmid that will reassemble into a circle.

After digestion with __________.

1. SalI alone
2. SalI and XhoI
3. SalI and PstI
4. SalI and SmaI
5. SmaI and PstI

B. In which of the treatments can the plasmid re-form into a circle by the
addition of DNA ligase after treating the cut DNA with DNA polymerase
in a mixture containing the four deoxyribonucleotides? Again assume that
you are trying to get the larger portion of plasmid to reassemble into a
circle.

10-20 Which of the following statements about genomic DNA libraries is false?
(a) The larger the size of the fragments used to make the library, the fewer
colonies you will have to examine to find a clone that hybridizes to your
probe.
(b) The larger the size of the fragments used to make the library, the more
difficult it will be to find your gene of interest once you have identified a
clone that hybridizes to your probe.
(c) The larger the genome of the organism from which a library is derived, the
larger the fragments inserted into the vector will tend to be.
(d) The smaller the gene you are seeking, the more likely it is that the gene
will be found on a single clone.
10-21 A DNA library has been constructed by purifying chromosomal DNA from mice,
cutting the DNA with the restriction enzyme NotI, and inserting the fragments
into the NotI site of a plasmid vector. What information cannot be retrieved from
this library?
(a) gene regulatory sequences
(b) intron sequences
(c) sequences of the telomeres (the ends of the chromosomes)
(d) amino acid sequences of proteins

10-22 You want to design a DNA probe used for hybridization to isolate a clone from a
cDNA library. Which of the following statements about DNA probes is true?
(a) The shorter the DNA probe used to probe the library, the greater the
number of colonies to which the probe will hybridize.
(b) A DNA probe that contains sequences that span two exons is better suited
to the purpose than a DNA probe that only contains sequences from one
exon.
(c) A DNA probe that contains sequences immediately upstream of the DNA
that codes for the first methionine in the open reading frame will usually
not hybridize to clones in a cDNA library.
(d) Hybridization of a DNA probe to the plasmid of interest will permit the
detection of the clone of interest; labeling of the DNA probe is not
necessary.

10-23 You have the amino acid sequence of a protein and wish to search for the gene
encoding this protein in a DNA library. Using this protein sequence, you deduce a
particular DNA sequence that can encode this protein. Why is it unwise to use
only this DNA sequence you have deduced as the probe for isolating the gene
encoding your protein of interest from the DNA library?

10-24 What is the main reason for using a cDNA library rather than a genomic library to
isolate a human gene from which you wish to make large quantities of the human
protein in bacteria?

10-25 Some clones from cDNA libraries can have defects because of the way in which a
cDNA library is constructed. For each dilemma (A to D) listed below, indicate which of
the outcomes (1 to 4) you might encounter, and explain why. Outcomes may be used
more than once.
10-26 You have an oligonucleotide probe that hybridizes to part of gene A from a
eucaryotic cell. Indicate whether a cDNA library or a genomic DNA library will
be more appropriate for use in the following applications.
A. You want to study the promoter of a gene A.
B. Gene A encodes a tRNA and you wish to isolate a piece of DNA
containing the full-length sequence of the tRNA.
C. You discover that gene A is alternatively spliced and you want to see
which predicted alternative splice products the cell actually produces.
D. You want to find both gene A and the genes located near gene A on the
chromosome.
E. You want to express gene A in bacteria to produce lots of protein A.

Note: The following codon table is to be used for Problems Q10-27, Q10-28, Q10-40,
Q10-43, Q10-44, and Q10-46.

10-27 Which of the restriction nucleases listed below can potentially cleave a segment
of cDNA that encodes the peptide KIGDACF?

10-28 Your biochemist friend has isolated a protein he thinks is responsible for making
you feel sleepy. Since he knows you’re taking Cell Biology, he wants you to help
him isolate the gene encoding this protein. Unfortunately, because your friend
could only isolate small amounts of protein, he was only able to obtain three short
stretches of amino acid sequence from the protein:

(a) H-C-W-K-M
(b) R-S-L-L-S
(c) D-A-Q-W-Y
 For each of the three peptides above, you design a set of DNA oligonucleotide
probes that can be used to detect the gene in a library. Which of the three sets of
oligonucleotide probes would be preferable for screening a library? Explain.
(Refer to the codon table above Q10-27.)

10-29 Your friend has isolated a protein present in the cheek cells of all straight-A
seniors at your school. She says that this protein helps you remember everything
you read and therefore will help cut down on the number of hours needed to study
for exams. She sequences the protein, which she calls “geniuszyme”, and designs
a probe to isolate the gene that encodes it. To make sure she designed the probe
correctly, she consults with the company that cloned Factor VIII. They have
100% confidence that her probe will work. She also obtains a high-quality liver
cDNA library from the company and uses her probe to try to isolate the gene for
geniuszyme. Unfortunately, she is unable to isolate any clones.
A. What is the likely explanation for her failure?
B. Not to be discouraged, your friend has obtained some genomic DNA
isolated from the nuclei of liver cells and has made a genomic library from
that DNA. Do you expect she will succeed in isolating the geniuszyme
gene from this library? Why or why not?

10-30 Insulin is a small protein that regulates blood sugar level, and it is given by
injection to people who suffer from the disease diabetes. Diabetics once used
insulin purified from pig pancreas to control their diabetes. Give two reasons why
the drug companies that produce insulin wanted to clone the human insulin gene
to provide an alternative source of the hormone.

10-31 Which of the following statements about PCR is false?

(a) PCR uses a DNA polymerase from a thermophilic bacterium.
(b) PCR is particularly powerful because after each cycle of replication, there
is a linear increase in the amount of DNA available.
(c) For PCR, every round of replication is preceded by the denaturation of the
doubled-stranded DNA molecules.
(d) The PCR reaction will generate a pool of double-stranded DNA
molecules, most of which will have DNA from primers at the 5′ ends.

10-32 Why is a heat-stable DNA polymerase from a thermophilic bacterium (the Taq
polymerase) used in the polymerase chain reaction rather than a DNA polymerase
from E. coli or humans?

10-33 Which of the following limits the use of PCR to detect and isolate genes?
(a) The sequence at the beginning and end of the DNA to be amplified must
be known.
(b) It also produces large numbers of copies of sequences beyond the 5′ or 3′
end of the desired sequence.
(c) It cannot be used to amplify cDNAs or mRNAs.
 (d) It will amplify only sequences present in multiple copies in the DNA
sample.

10-34 You want to amplify the DNA between the two stretches of sequence shown in
Figure Q10-34. Of the listed primers, choose the pair that will allow you to
amplify the DNA by PCR.

Figure Q10-34

10-35 Your friend works at the Centers for Disease Control and has discovered a brand-
new virus that has recently been introduced into the human population. She has
just developed a new assay that allows her to detect the virus by using PCR
products made from the blood of infected patients. The assay uses primers in the
PCR reaction that hybridize to sequences in the viral genome.

Your friend is distraught because of the result she obtained (see Figure Q10-35)
when she looked at PCR products made using the blood of three patients suffering
from the viral disease, using her own blood, and using a leaf from her petunia
plant.

You advise your friend not to panic, as you believe she is missing an important
control. Which one of the choices listed below is the best control for clarifying the
results of her assay? Explain your answer.

Figure Q10-35

(a) a PCR reaction using blood from a patient who is newly infected but does
not yet show symptoms
(b) a PCR reaction using blood from a dog
 (c) a PCR reaction using blood from an uninfected person
(d) repeating the experiments she has already done with a new tube of
polymerase

Deciphering and Exploiting Genetic Information

10-36 For each of the following sentences, fill in the blanks with the best word or phrase
selected from the list below. Not all words or phrases will be used; each word or
phrase should be used only once.

The technique of __________________ hybridization can be used

to detect a specific RNA expression in a particular region of the
brain. Northern blotting detects a specific sequence in
__________________. Southern blotting detects a specific
sequence in __________________. A short, single-stranded DNA
is a(n) __________________. A piece of DNA used to detect a
specific sequence in a nucleic acid by hybridization is known as
a(n) __________________.

DNA polymerase chain reaction

in situ probe
in vitro RNA
oligonucleotide vector

10-37 In situ hybridization can be used to determine the _________________.

(a) sequence of a cloned gene
(b) distribution of proteins in tissues
(c) size of a gene
(d) distribution of a given type of mRNA in different tissues

10-38 Why are dideoxyribonucleoside triphosphates used during DNA sequencing?

(a) They cannot be incorporated into DNA by DNA polymerase.
(b) They are incorporated into DNA particularly well by DNA polymerases
from thermophilic bacteria.
(c) Incorporation of a didoxyribonucleoside triphosphate leads to the
termination of replication for that strand.
(d) Dideoxyribonucleotide triphosphates are more stable than
deoxyribonucleoside triphosphates.

10-39 Which of the following describes a feature found in bacterial expression vectors
but not in cloning vectors?
(a) origin of replication
(b) cleavage sites for restriction nucleases
(c) promoter DNA sequences
(d) a polyadenylation signal
10-40 You have sequenced a short piece of DNA and produced the gel shown in Figure
Q10-40.

Figure Q10-40

A. What is the sequence of the DNA, starting from the 5′ end?

B. If you knew that this sequence is from the middle of a protein-coding
cDNA clone, what amino acid sequence can be deduced from this
sequence? (Refer to the codon table above Q10-27.)

10-41 You have sequenced a fragment of DNA and produced the gel shown in Figure
Q10-41. Near the top of the gel, there is a section where there are bands in all four
lanes (indicated by the arrow). Which of the following mishaps would account for
this phenomenon? Explain your answer.

Figure Q10-41
 (a) You mistakenly added all four dideoxynucleotides to one of the reactions.
(b) You forgot to add deoxynucleotides to the reactions.
(c) Your primer hybridizes to more than one area of the fragment of DNA you
are sequencing.
(d) A restriction nuclease cut a fraction of the DNA you are sequencing.

10-42 You are interested in understanding the gene regulation of a protein called LKP1,
which is normally produced in liver and kidney cells in mice. Interestingly, you
find that LKP1 is not expressed in heart cells. You replace the coding sequence
for LKP1 with the DNA encoding green fluorescent protein (GFP) and examine
the expression of GFP in mice. (GFP is your reporter gene.) You find expression
of GFP in liver and kidney cells but not heart cells; this expression pattern is
similar to how LKP1 is expressed normally. Further experiments demonstrate that
there are three regulatory sequences in the promoter, labeled A, B, and C in
Figure Q10-42, that are important for this expression pattern. You want to
determine the significance of each regulatory sequence, so you create situations
where only a subset of regulatory regions are present upstream of the reporter
gene, as diagrammed in Figure Q10-42.

Figure Q10-42

Given the data, which of the statements below is false?

(a) Regulatory sequence A switches on LKP1 in the kidney.
(b) Regulatory sequence B switches on LKP1 in the liver.
(c) Neither regulatory sequence A nor regulatory sequence B is required for
LKP1 expression in the heart.
(d) Regulatory sequence C is not necessary for proper expression.
10-43 You are studying a protein, and a small fragment of its sequence is shown below.
You have decided that the glutamine in the protein segment has an important role
in its function. You decide to change this glutamine to a lysine using DNA
technology with the use of site-directed mutagenesis. You have a plasmid that
contains the full-length version of the gene that encodes this protein and wish to
create a new DNA molecule that has a change in only one base and that
substitutes a lysine for a glutamine. Given the partial mRNA sequence that codes
for this stretch of protein below, devise a 21-nucleotide DNA oligonucleotide that
can be used to make this mutation. Be sure to label the 5′ and 3′ ends. (Refer to
the codon table above Q10-27.)

F D P Q G S H
5′-uucgacccgcagggcagccac–3′

10-44 You are studying a protein that contains the peptide sequence RDWKLVI. The
part of the DNA encoding this peptide is included in the sequence shown below.

5′-GGCGTGACTGGAAGCTAGTCATC-3′
3′-CCGCACTGACCTTCGATCAGTAG-5′

This sequence does not contain any HindIII restriction enzyme sites; the target
sequence for the HindIII restriction nuclease is shown in Figure Q10-44.

Figure Q10-44

Your goal is to create a HindIII site on this plasmid without changing the coding
sequence of the protein. Explain how you would do this. (Refer to the codon
table above Q10-27.)

10-45 You are interested in understanding how the brain works, and are using the fruit
fly Drosophila as a model system to study brain development. You perform a
microarray analysis to try to determine genes expressed in the fly brain. For your
microarray experiment, you first prepare cDNA from fly brains and label it with a
red fluorochrome. Then you isolate cDNA from whole flies and label it with a
green fluorochrome. Next you hybridize these cDNA populations to a microarray
containing the Drosophila genes. From this you obtain a list of genes that are
specifically enriched in the brain (those that show up as a red spot on the
microarray).

You are disappointed because your favorite fly gene, tubby, does not appear on
this list, even though you have repeated the microarray experiment 10 times and
did not encounter any technical difficulties. The reason you thought tubby would
appear on this list is that you believe tubby is important for brain development,
because flies mutant in tubby have no brains. Not to be discouraged, you perform
 in situ analysis using the tubby DNA as a probe, and see that it is expressed at
high levels in the fly brain of normal flies but not expressed in animals lacking the
tubby gene.

Why do you think tubby did not show up as a gene specifically enriched in the
brain in your microarray experiment?

10-46 You have created a piece of recombinant DNA by placing a cDNA from a gene
you believe is important for the differentiation of liver cells (called LC1) onto an
expression plasmid that contains all the sequences necessary for propagation of
this DNA in bacteria and for the production of the LC1 protein in bacteria. A
picture of this plasmid is shown in Figure Q10-46A, with the segment of the DNA
containing the PK1 gene depicted as a grey rectangle; the promoter sequence is
depicted as a white rectangle. The LC1 protein is phosphorylated on serine 54; the
nucleotide sequence of the portion of the DNA that encodes this region is shown
below. All HindIII and SalI restriction sites have also been mapped on the
plasmid; the recognition sequences for these restriction nucleases are shown in
Figure Q10-46B.

Figure Q10-46

A. Given the information above, write out the amino acids 52 to 57, encoded
by the nucleotide sequence shown above. Be sure to number the amino
acids appropriately. (Hint: remember, serine is amino acid number 54.)
(Refer to the codon table above Q10-27.)
B. You want to create a mutant version of the PK1 gene that replaces serine
54 found on this peptide with a glycine. You want to do this by changing
only one nucleotide, and you also want to destroy the HindIII recognition
sequence with this change. Write out a 21-nucleotide DNA primer that can
be used for site directed mutagenesis to accomplish this task. Be sure to (i)
write out the DNA and label the 5′ and 3′ ends, (ii) underline the mutated
HindIII recognition site, and (iii) circle any change made to the original
sequence.

10-47 Which of the following statements about RNA interference (or RNAi) is false?
(a) RNAi is a natural mechanism used to regulate genes.
 (b) During the process of RNAi, hybridization of a small RNA molecule with
the mRNA degrades the mRNA.
(c) Because RNAi depends on the introduction of a double-stranded RNA into
a cell or an organism, it is not a process that can cause heritable changes in
gene expression.
(d) In C. elegans, RNAi can be introduced into the animals by feeding them
with bacteria that produce the inhibitory RNA molecules.

10-48 You have been hired to create a cat that will not cause allergic reactions in cat-
lovers. Your co-workers have cloned the gene encoding a protein found in cat
saliva, expressed the protein in bacteria, and shown that it causes violent allergic
reactions in people. But you soon realize that even if you succeed in making a
knockout cat lacking this gene, anyone who buys one will easily be able to make
more hypoallergenic cats just by breeding them. Which of the following will
ensure that people will always have to buy their hypoallergenic cats from you?
(a) Inject the modified ES cells into embryos that have a genetic defect to
prevent the mature adult from reproducing.
(b) Implant the injected embryos into a female cat that is sterile as a result of a
genetic defect.
(c) Sell only the offspring from the first litter of the female cat implanted with
the injected embryos.
(d) Surgically remove the sexual organs of all the knockouts before you sell
them.

How We Know: Sequencing the Human Genome

10-49 You have been asked to consult for a biotech company that is seeking to
understand why some fungi can live in very extreme environments, such as the
high temperatures inside naturally occurring hot springs. The company has
isolated two different fungal species, F. cattoriae and W. gravinius, both of which
can grow at temperatures exceeding 95°C. The company has determined the
following things about these fungal species:

By sequencing and examining their genomes, the biotech company hopes to

understand why these species can live in extreme environments. However, the
company only has the resources to sequence one genome, and would like your
input as to which species should be sequenced and whether you believe a shotgun
strategy will work in this case. (Be sure to explain your answer.)

10-50 Figure Q10-50A depicts the restriction map of one segment of the human genome
for four restriction nucleases W, X, Y, and Z. Figure Q10-50B depicts the
restriction maps of four individual BAC clones that contain segments of human
DNA from the region depicted in Figure Q10-50A.

Figure Q10-50

From this information, how would you order these BAC clones, from left to right?
(a) 1, 2, 3, 4
(b) 2, 1, 4, 3
(c) 3, 4, 2, 1
(d) 4, 1, 3, 2
Answers

10-1 (c)

10-2 (b). Restriction nucleases cleave double-stranded DNA.

10-3 (c). A restriction nuclease that has a 4-base-pair recognition sequence cuts on
average once every 44 or 256 base pairs; one that has a 6-base-pair recognition
sequence cuts once every 46 or 4096 base pairs; one that has an 8-base-pair
recognition sequence cuts once every 48 or 65,536 base pairs; one that has a 12-
base-pair recognition sequence cuts once every 412 or 16 million base pairs. Thus,
to obtain fragments of about 70 kb in size, you would cut with a nuclease that
recognizes an 8-base-pair site.

10-4 (b)

10-5 (d)

10-6 A. You would first digest your sample with a combination of restriction
enzymes selected so that they give a set of fragment sizes that could have
come from only one of the plasmids. Then you would run the resulting
mixture of DNA fragments on a gel alongside a set of size markers and
determine the size of each fragment. By looking at the restriction maps,
you should then be able to match your results to one of the plasmids.
B. Digestion with any of the following combinations will enable you to
distinguish which plasmid you have: HindIII + BglII; EcoRI + BglII;
EcoRI + BglII + HindIII. The plasmids are the same size, so you cannot
distinguish between them simply by making a single cut (with HindIII)
and determining the size of the complete DNA by gel electrophoresis. Nor
can you distinguish them by cutting with all four restriction nucleases,
Because the set of fragment sizes produced from both plasmids will be the
same. Cutting with BamHI or EcoRI on their own is not sufficient because
you will get bands of the same size from both plasmid A and plasmid B.
The only difference between the two plasmids is in the location of the
BglII site relative to the two BamHI sites, so if you cut with an enzyme
that cuts outside the BamHI fragment and with BglII, you will get
different-sized fragments from the two plasmids.

10-7 A. All three are affected.

B. The two parents have a single defective copy of the gene; the fetus has two
defective copies.
C. As seen in the two blots in Figure Q10-7B, Caril Ann and Charles each
have one full-length copy of gene X (the bands at the top of the gel),
which hybridizes with both probe A and probe B. The fetus does not. The
blot with probe A shows that Caril Ann and the fetus have a short
fragment of gene X that hybridizes with probe A only, indicating that this
 copy of gene X has a deletion somewhere other than in the region
recognized by probe A. The second blot (with probe B) shows that Charles
and the fetus have a short fragment that hybridizes with probe B,
indicating that this copy of gene X has a deletion somewhere other than in
the region recognized by probe B. Because the shortened gene found in
Charles does not show up on the probe A blot, this deletion must be in the
region of A; similarly, because the shortened gene found in Caril Ann
does not show up on the probe B blot, her deletion must be in the region of
B. The fetus has inherited two defective copies of gene X, one from each
parent.

10-8 A. All four oligonucleotide probes.

B. Oligonucleotide probe B and oligonucleotide probe D. Both the upper and
lower strands of DNA are present in genomic Southern blots, so all four
oligonucleotides will hybridize to either Southern blot. (Oligonucleotides
A and B will still be able to hybridize to genomic DNA cut with BglII,
because they can still base-pair with the individual fragments that result
from the digest.) Northern blots contain only RNA, which has the
sequence of the upper strand of the DNA. Hence, only oligonucleotide
probe B and oligonucleotide probe D will hybridize to a Northern blot.

10-9 (a)

10-10 (c)

10-11 (d)

10-12 A nuclease hydrolyzes the phosphodiester bonds in a nucleic acid. Nucleases that
cut DNA only at specific short sequences are known as restriction nucleases.
DNA composed of sequences from different sources is known as recombinant
DNA. Gel electrophoresis can be used to separate DNA fragments of different
sizes. Millions of copies of a DNA sequence can be made entirely in vitro by the
polymerase chain reaction technique.

10-13 Two fragments of DNA can be joined together by DNA ligase. Restriction
enzymes that cut DNA straight across the double helix produce fragments of
DNA with blunt ends. A fragment of DNA is inserted into a vector in order to be
cloned in bacteria. A cDNA library contains a collection of DNA clones derived
from mRNAs. A genomic library contains a collection of DNA clones derived
from chromosomal DNA.

10-14 A cloning vector for use in bacteria must contain the following:
1. a bacterial replication origin (to allow the plasmid to be replicated)
2. at least one unique restriction site (to allow easy insertion of foreign DNA)
3. an antibiotic-resistance gene or some other selectable marker gene (to
allow selection for bacteria that have taken up the recombinant plasmids)
Another random document with
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escondida,
la causa no sufre poder
publicarse,
ni para decirse ni para callarse
ni entrada se halla, ni tiene
salida.
Mudar ni oluidar ya no es en
mi mano,
ni puede quererse ni puedo
querello,
porque el menor daño está en
padezello
y en mí lo doliente es mejor
que lo sano;
es grande el dolor, mas es tan
ufano
que veo perderse mi vida de
claro,
si más no perdiesse no es
mucho ni caro
que cierto en perdella
perdiendo la gano.
El fuego que dentro del
alma m'abrasa
su pena es tan graue que no
sé dezilla,
querria viuir por solo sofrilla
mas este querer la muerte me
acusa;
conoze en mis males que no
se m'escusa,
pues toda la causa está en mi
desseo,
más mal no pudiera hacerme
Perseo
aunque me mostrara la faz de
Medusa.
 (Habla contra el amor.)
Contentate agora, amor
engañoso,
pues todos tus fuegos con
tanto furor
encienden y abrasan de vn
pobre pastor
sus tristes entrañas, sin dalle
reposo:
bien te podrás llamar vitorioso
venciendo vn vencido que
quiso vencerse
de quien imposible le fue
defenderse
ni tú si le viesses serias
poderoso.
Esfuerça tus fuerças en mí
pobrecillo,
enciende con ellas mi fuego
mortal,
que quanto más creces la
pena en mi mal
la causa me hace contento
sofrillo;
empleas tus flechas en vn
pastorcillo
rustico, solo de bien y de
abrigo,
que no podrán tanto tus
mañas comigo
que desto m'apartes, ni menos
dezillo.

(Habla con su soledad.)

 Venid soledad, leal
compañia,
que solo con vos me hallo
contento,
con vos gozo más de mi
pensamiento
que nunca se parte de mi
fantasia,
vos no me dexais, dexóme
alegria,
plazer ni esperança en quien
ya no espero,
reposo, descanso, tampoco
los quiero
ni nada de quanto primero
tenia.

(Habla al ganado.)
O triste ganado qu'estás sin
señor
a solas paciendo, pues solo te
dexo,
quexarte has de mí, tambien
yo me quexo
del mal que sin culpa me haz'
el amor.
No plangas perder tan triste
pastor
de quien no esperabas ya
buena pastura,
pues él ya no espera sino
desuentura,
dexalo a solas passar su dolor.
E vos mi çurron, e vos mi
rabel
que soys el descanso que
traygo comigo,
pues veys que me veo quedar
sin abrigo,
razon es que quede sin vos e
sin él;
n'os duela partir agora d'aquel
que hasta el morir aun dél se
desdeña,
e vos mi cuchar e vos mi
barreña
andayos con dios, partios
tambien dél.
A solas quedad comigo,
cayado,
pues todo lo dexo y pasar no
me dexa,
al menos con vos del mal que
m'aquexa
podré sostenerme estando
cansado;
dexé mi çurron, rabel e
ganado,
la yesca, eslabon, barreña,
cuchar,
dexé mis plazeres, mas no mi
pesar
e menos a vos tampoco he
dexado.
Agora reposo que solo me
veo,
agora descanso en medio mis
males,
o lagrimas mias, o ansias
mortales,
o tristes sospiros con quien yo
peleo;
la vida aborrezco, la muerte no
veo,
que aun essa me niega su
triste venir,
e trueca el matarme con
darme el viuir
por no complazer mi triste
desseo.
O más aborrido pastor sin
ventura
de quantos oy viuen en toda la
tierra,
nin todo lo llano, nin toda la
sierra
nin todos los bosques, ni otra
espesura;
quien t'a de sanar, tu muerte
procura,
no tienes reparo, ni tienes
abrigo,
ni tienes pariente, ni tienes
amigo,
si mueres te falta tambien
sepultura.
Agora estaras, Torino,
contento
que tú de tu mano te diste
herida
que basta quitarte mill vezes la
vida
sola la causa de tu
pensamiento,
medido do llega su
merecimiento
vista tu suerte quedar tan
atrás
que quieres tu pena y no
quieres más
y no te consienten sofrir tu
tormento.
¿Dónde toviste, Torino, el
sentido,
cómo podiste tan presto
perdello?
¿que vees tu mal, no pues no
querello?
si quexas, tus quexas no eres
oydo,
consientes tu mal e no eres
creydo.
Mejor te seria del todo morir
que verte penando muriendo
seruir
do solo es tu pago tenerte
aborrido.
Oido yo a huego quexuras
tamañas
como este pastor descubre
que siente,
yo nunca vi en otro qu'estando
doliente
dixese que s'arden en él sus
entrañas;
yo creo que tiene heridas
extrañas
que quieren del todo con
yerua matallo,
quiero buscar quien venga a
curallo
si puedo hallarlo por estas
cabañas.
Quiça l'a mordido perro
dañado
o qualq'animal o lobo rabioso
pues da tales buelcos, no
tiene reposo
y esta delos ojos ciego
turbado;
no vee do dexa çurron ni
cayado,
vertida la yesca, quebrado el
rabel,
o es el demoño que anda con
él
o qualque desastre que tiene
el ganado.
O si con su amo quiça si ha
reñido
si quiere lleualle qualque
meçada,
mas él no haria por poca
soldada
estandose a solas tamaño
roydo;
miafe que pienso que no es so
mordido,
c'aquellos solloços no son de
buen rancho,
quiero traballe del pie con el
gancho,
quiça si lo sueña estando
adormido.

(Habla el mismo Guillardo

admirandose
porque no le sintio trauando
del.)
O dolo a dios y
cómo no siente?
mayor es que sueño
este su mal,
 alli me pareze que
viene Quiral
que le es gran amigo
y aun cabo pariente,
quiero llamallo, zagal
es valiente,
oyes, Quiral, allegate
acá.
Q. Miafe, Guillardo,
yo ya me yua allá
que bien ha buen rato
que lo tengo en miente.
G. Pues yo te he
llamado por fazer tu ruego
que vengas a ver tu
amigo Torino,
que aqui le he
hallado tan fuera de tino
que dize que s'arde
en brasas de fuego.
Q. Quiça habra
perdido o choto o borrego
y está maldiziendo la
res que lo cria.
G. No es esse el mal,
Quiral, que dezia,
mayor es el daño de
qu'él está ciego.
Yo me he
quillotrado tan junto con él
que de las manos le
quité el cayado,
ni él me sintio ni mira
al ganado,
ni cura si andan los
lobos en él;
 acá está el çurron,
allá está el rabel,
y el no son sospiros y
ahuncos de muerte
diziendo y quexando
su mal qu'es tan fuerte
que passa los otros
de pena cruel.
Y aun tengo
sospecha quiça qu'está
enfermo
según l'he sentido tan
gran comezon,
que deue tomalle
qualque torozon
d'andar passeando
de noche este yermo.
Q. Miafe, pues vamos
a vello, Guillermo,
pues sabes la via, da
tú camino.
G. Helo aqui está
debaxo este pino.
Q. Duermes, Torino?
T. ¿Que qués, que
no duermo?
Q. Pues saluete Dios.
T. Vengais
norabuena.
Q. Qué sientes,
Torino, que gimes tan fuerte?
T. Siento, pastores, el
mal de la muerte
y essa no llega por
darme mas pena;
passion me combate,
razon me condena,
 dolor me fatiga,
tristeça me aquexa,
querria sanar, querer
no me dexa,
los males son mios,
la causa es agena.
Q. Yo creo que tienes
esprito malino,
per signum crucis a
dios recomiendo,
ni sé lo que dizes ni
menos t'entiendo,
harasme dezir que
hablas con vino.
Retorna, retorna,
retorna, Torino,
razona con tiento,
con seso y de vero,
peor seras tú que
Juan Citolero
con sus patrañuelas
que s'anda contino.
T. No te marauilles
m'abraso en inuierno
y enmedio el verano
perezco de frio,
no he visto otro mal
assi como el mio
y assi le juzgo de
todos moderno.
Q. Date, Torino, date
gobierno,
si aqui no estás sano
muda majada.
T. Primero, Quiral,
por medio el yjada
 mi mal reuiente y se
vaya al infierno.
Q. ¿Qué mal puede
ser tan crudo que sientas
lo mucho que duele y
callas tu fatiga?
¿es mal dellonbrigo o
dolor de barriga
que dices el daño y la
causa no cuentas?
Veo en ti dolor que
revientas,
¿es mal de costado
que a todos avança?[287]
T. No es esse, Quiral,
es poca esperança,
qu'es muy mas cruel
que cuanto me mientas.
Q. ¿De qué
desesperas? ¿has algo sembrado
que piensas perdello
o quiça que no naça,
o has miedo que falte
lugar donde paça
en estos exidos tu
poco ganado?
T. No es este, pastor,
mi graue cuydado,
mas verme penado e
de muerte herido
de mano de quien me
tiene aborrido
y assi desespero de
ser remediado.
Q. Ahotas que pienso
que tu mal oteo
 e dudo que creo
qu'es mal d'amorio,
dalo al demoño tan
gran desuario
que mata la vida su
solo desseo.
T. Mayor es el daño,
Quiral, que posseo
qu'en todos los males
que sufro e consiento
fallece esperaba e
crece tormento
y en todos los medios
remedio no veo.
Q. Do yo al demoño
la hembra maldita
que mata un zagal
assi de passion.
T. Calla, Quiral, por
Dios tal razon
que solo en oyllo la
vida me quita,
que no es quél tú
dizes mas antes bendita
segun las virtudes
que caben en ella.
Q. ¿Pues cómo la
alabas y quexaste della?
Dime quien es, quiça
si es Benita.
La nieta d'aquel
que hu mayoral
de todos los hatos
d'aquesta dehesa
y hija d'aquel que con
justa empresa
 teniendo justicia
perdió tribunal,
y aun hija d'aquella
que dizen qu'es tal
qu'en todas las otras
que viuen agora
ninguna se halla tan
noble señora
que sea con ella en
nobleça ygual.
Pues si esta que
digo tanto es hermosa
que basta alegrarte
con su fermosura
e basta a dar vida a
qualquer criatura
e mas como dizes
qu'es tan virtuosa,
pues date reposo,
reposa, reposa,
si assi como dizes
tan fuerte la quieres,
siendo ella tal, dime
porqué mueres,
siendo tu llaga en si
gloriosa?
T. Yo no sé dezir el
mal de que muero
ni tú lo sabrias
podiendo sentillo,
yo sélo sentir mas no
sé dezillo,
ni sé lo que pido ni sé
lo que quiero,
socuños termeños, te
digo de vero
 que tiene quien vella
d'amor me condena,
tornando a miralla me
crece más pena
que dexame siempre
más mal que primero.
Q. Plazer me daria si
yo de ti fuesse.
T. Dolo al demoño,
Quiral, tu consejo,
diran que vi en ella
algun aparejo
por do mi esperança
esperança tuuiesse,
y aun más me diria
quien tal en mi viesse
que ando perdido sin
seso y sin tiento
pues saben qu'es
tanto su merecimiento,
qu'es poco mi mal si
dél yo muriesse.
Q. Miafe, pues
quedate con tu dolor
pues tú te lo quieres
y quexas tu mal.
T. Querria una cosa
tan solo, Quiral,
que fuese tan grande
qual es e mayor
con que Benita
mostrasse color,
qu'es ella contenta
que yo lo sufriesse;
si esto, Quiral, Benita
hiziesse
 jamas pediria más
bien ni favor.
G. Di que t'a dicho
por tu fe, Quiral,
¿qué dolor siente que
assi lo apollina?
¿Tienes tú huzia que
haura melecina
o asmo que pienso
qu'es gota coral?
Q. Miafe, Guillardo,
su mal es un mal
c'allá do se sienta por
mal de pecados
harto mal año y pro
malos hados
tien el pastor que se
pone en lo tal.
G. ¿Qué mal puede
ser c'así percudia
y assi lo ahuncava
con tanto cariño
que daua chillidos
assi como un niño
que no parecia so
que se moria?
Q. Un mal es,
Guillardo, de tanta porfia
qu'es bien de plañir
aquel q'el acude.
G. Dolo al demoño y
tan fuerte percude
que no da reposo ni
noche ni dia.
Q. Un mal es que
s'entra por medio los ojos
 e vase derecho hasta
el corazon,
alli en ser llegado se
torna afficion
e da mil pesares,
plazeres y enojos,
causa alegrías,
tristeças, antojos,
haze llorar y haze
reyr,
haze cantar y haze
plañir,
da pensamientos dos
mill a manojos.
G. ¿Es biuora o qué
o es alacran
o es escorpion, o es
basilisco,
que yo oy dezir aquí
en nuestro aprisco
que a todos los mata
los qu'á velle van?
Q. Amor es,
Guillardo, que da mas afan
de pena crecida y
ansiosas fatigas.
G. Daldo al demoño,
hartaldo de migas,
dalde cuajada e
queso y aun pan.
Si fruta quisiere dalde
castañas,
dalde mançanas,
vellotas, piñones.
Q. No come Guillardo
sino corazones
 y higados viuos y
viuas entrañas.
G. Echaldo de fuera
de vuestras cabañas
a ese demoño
gusano cruel.
Q. Miafe, no valen
sañas con él
ni valen razones ni
fuerças ni mañas.
G. ¿Pues cómo se
sana quillotro tan fuerte?
dalde triaça, yo la
traygo en mi esquero.
Q. No es buena,
modorro, que si es verdadero
no tiene salud jamas
sin la muerte.
G. Pues si ese diabro
es mal dessa suerte,
segun que yo veo
morir so Torino.
Q. Morir si me dizes,
ya muere el mezquino,
¿no vees que su vida
en morir se convierte?
G. O dome a dios y a
san Berrion,
si vello pudiesse,
Dios me confonda
si no le matasse con
esta mi honda
porque él no matasse
assi esse garçon.
Q. Calla, bestiazo,
que no anda en vision
 para que puedas assi
dalle empacho.
G. O dolo al fuego,
¿es hembra o es macho,
o es duen de casa o
qualque abejon?
Q. Es cosa que nace
de la fantasia,
y ponese enmedio
dela voluntad,
su causa primera
produze beldad,
la vista la engendra el
corazon la cria,
sostienela viua
penosa porfia,
dale salud dudosa
esperança,
si tal es qual deue no
haze mudança,
ni alli donde está
nunca entra alegria.
G. O yo no t'entiendo
o no sé que s'es,
ni es esso ni essotro,
ni es cosa ni al,
tú dizes qu'es bien, tú
dizes qu'es mal,
no es bestia, ni es
ave, ni pece, ni es res,
no está del derecho
ni está del enues,
no dexa viuir, ni mata
tampoco,
no es gusarapa, no
es cuerdo ni loco;
 pues yo te prometo
que a la fin algo es.
Mas helo aqui
torna Torino turbado,
con su mortalera de
rabia o cordojo,
quiero pedille si es
fiebre o enojo
y hazer que lo diga
por fuerça o de grado.
Dime, Torino, qué mal
t'a tomado
que assina te trae
desaborrecido,
ca este demoño
jamas l'entendido
mill desbariones
c'aquí m'a contado.
T. Guillardo,
Guillardo, mi mal es c'adoro
d'amor a Benita
porqu'es mi señora,
mi vida la quiere, mi
alma l'adora
y ella me trata peor
que a un moro.
G. O dom'a dios e
agora lo yñoro,
esso que dizes
querencia se llama,
quando un zagal dize
que ama,
yo ya lo sabia, miafe,
de coro.
Tú andas, Quiral,
chuchurreando
 con chichorrerias en
chicharramanchas,
en prietas, en
blancas, en cortas y en
anchas,
y no me quillotras lo
que te demando,
¿qué te calle andar
quillotrando
del mal que a Torino
le daua porfia?
que aunque no lo sé
yo ya lo sabia
qu'es una locura que
s'anda burlando.
Y di, tú, Torino,
qu'eres sabiondo
¿assi te percossas
por una zagala?
haue verguença de ti
noramala,
no digan que eres
algun berriondo.
T. Guillardo,
Guillardo, mi mal es tan hondo
que no puedo ya ni
quiero valerme,
si hallo remedio con
que defenderme
aquel es el mismo
con que me confondo.
G. Pues hela aqui
viene, laque assi te mata,
con otra zagala que
se anda tras ella,
levanta, Torino, e
vamos a ella